Technical Abstract:
Within the last few years, outbreaks of highly pathogenic avian influenza (HPAI) have originated in Asia and spread through several Middle Eastern, African and European countries, resulting in one of the most serious animal disease incident in recent history. These outbreaks were characterized by the unusual pathogenicity of these viruses in wild birds, with some isolates capable of causing significant morbidity and mortality in wild birds and mammals. To better understand the mechanisms of pathogenesis of these HPAIV in poultry we have infected chickens with the Egret/Hong Kong/757.2/02 isolate of H5N1 HPAIV. RNA expression levels from lung and spleen tissue were compared to mock infected and vaccinated infected chickens using a whole genome (44K genes) chicken microarray. A procedure to obtain high quality tissue RNA followed by dual color labeling and hybridization was optimized and used to compare changes in gene expression. GeneSpring 7.0 computer software (Agilent) was used for all normalization and statistical analysis. Lungs and spleens from six birds were compared for each experimental condition and genes with statistically significant differences identified. Evidence of a pronounced inflammatory response that resembled mammalian cytokine responses was observed in infected birds. In contrast, this inflammatory response was absent in vaccinated birds. An involvement of multiple inducers and responders of the interferon pathway was evident. Semi-quantitative RRT-PCR analysis was used to confirm expression at the RNA levels, and immunohistochemistry confirmed expression of critical genes at the protein level.